Optimization of Culture Conditions and Continuous Production of ...

Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 1 9Table 2. Comparison of the results of two previous trials of continuous culture with the present workDavoust and Hansson [9] Rane and Hoover [10] This workStrain Absidia coerulea Absidia coerulea Absidia coeruleaCarbon Source Glucose 20 g/L Glucose 20 g/L Glucose 20 g/LNitrogen SourceYeast extract 6 g/L(NH 4) 2SO 411 g/LYeast extract 1 g/LPeptone 10 g/L(NH 4) 2SO 45 g/LYeast extract 15 g/L(NH 4) 2SO 45 g/LSaltsKH 2PO 43 g/LMgSO 40.6 g/LZnSO 4⋅ 7H 2O 1.8 mg/LMnSO 4⋅ H 2O 0.3 mg/LCuSO 4⋅ 5H 2O 0.4 mg/LK 2HPO 41 g/LNaCl 1 g/LMgSO 4⋅ 7H 2O 5 g/LCaCl 20.1 g/LK 2HPO 41 g/LNaCl 1 g/LMgSO 4⋅ 7H 2O 5 g/LCaCl 20.1 g/LAdditive Fe-EDTA 1.3 g/L - -Dilution Rate (h -1 ) 0.03-0.15 0.025 0.05Agitator Speed (rpm) 300 150 250Result Failure Success SuccessChitosan Concentration (g/L) - 1.36 1.04Percentage of Chitosan in the Cell (%) - 10.9 14.0Chitosan Productivity (g L -1 h -1 ) - 0.034 0.052Granule-like spores and mycelia were observed in thebroth during continuous culture. This broth could beused as the seed, assured it formed pellets and itsgrowth was like that obtained using a spore suspension.Our results emphasize that it is very important to controlthe medium composition for the continuous cultureof Absidia coerulea, to enable the formation of newgrowth spots.CONCLUSIONCell growth and chitosan production are highly influencedby culture conditions such as pH, agitator speed,and aeration rate. Optimum conditions obtained for thebatch cultivation of Absidia coerulea were a pH 4.5, anagitator speed of 250 rpm, and an aeration rate of 2 vvm.The maximum specific growth rate and chitosan concentrationobtained under these conditions were 0.08 h -1and 2.3 g/L, respectively.Continuous culture was undertaken to increase productivityand produce chitosan with unique properties.The pellet structure was found to be very important forthe formation of new spots in continuous culture andthis was influenced by medium composition and cultureconditions. Chitosan productivity obtained in thisstudy was 0.052 g L -1 h -1 . Because few difficulties wereencountered during the operation, continuous culturewith Absidia coerulea ATCC 14076 was carried out successfullyand proved to be effective at producing chitosanwith high productivity.Acknowledgements This work was supported by theBrain Korea 21 Project.REFERENCES[1] Allan, G. G., J. R. Fox, and N. Kong (1978) A criticalevaluation of the potential source of chitin and chitosan.pp. 64-78. In: R. A. A. Muzzarelli and E. R. Pariser (eds.).Processings of the First International Conference on Chitin/Chitosan.MIT sea grant report MITSG 78-7, Index no.78-307-Dmb. Massachusetts Institute of Technology,Cambridge, USA.[2] Muzzarelli, R. A. A. (1997) Chitin. Pergamon Press, Oxford,UK.[3] Bartnicki-Garcia, S. (1968) Cell wall chemistry, morphogenesisand taxonomy of fungi. Annu. Rev. Microbiol. 22:87-108.[4] White, S. A., P. R. Farina, and I. Fultons (1979) Productionand isolation of chitosan from Mucor rouxii. Appl.Environ. Microbiol. 38: 323-328.[5] MaGahren, W. J., G. A. Perkinson, J. A. Growich, R. A.Leese, and G. A. Ellestad (1984) Chitosan by fermentation.Process Biochem. 19: 88-90.[6] Shimahara, K. and Y. Takiguichi (1988) Preparation ofcrustacean chitin. Method Enzymol. 161: 417-423.[7] Shimarahara, K., Y. Takaguichi, T. Kobayachi, K. Uda,and T. Sannan (1989) Screening on mucoraceae strainssituable for chitosan production. pp. 171-178. In T.Skjäk-Bræk, T. Anthonsen, and P. Sandford (eds.) Chitinand Chitosan. Elsevier, London, UK.[8] Hang, Y. D. (1990) Chitosan production from Rhizopusoryzae mycelia. Biotechnol. Lett. 12: 911-912.[9] Davoust, N. and G. Hansson (1992) Idenfying the conditionsfor development of beneficial mycelium morphologyfor chitosan-producing Absidia spp. in submersedcultures. Appl. Microbiol. Biotechnol. 36: 618-620.[10] Rane, K. D. and D. G. Hoover (1993) Production of chito-